Genes for male accessory gland proteins in Drosophila melanogaster

ABSTRACT

The present invention provides a number of accessory gland proteins from  Drosophila . The invention also provides an accessory gland protein which is toxic to insect cells and can be used to kill or inhibit the development of insects. Methods of controlling insects are also provided.

The present application is a divisional of U.S. patent application Ser.No. 10/114,774, filed Apr. 4, 2002, now U.S. Pat. No. 6,955,897, issuedOct. 18, 2005, which is a divisional of U.S. patent application Ser. No.09/219,983, filed Dec. 23, 1998, now U.S. Pat. No. 6,380,159, issuedApr. 30, 2002, which claims the benefit of U.S. Provisional PatentApplication Ser. No. 60/071,315, filed Dec. 23, 1997.

The subject matter of this application was made with support from theUnited States Government under Grant Nos. IBN97-23356, IBN94-06171, andDCB91-08221 from the National Science Foundation. The United StatesGovernment may retain certain rights.

BACKGROUND OF THE INVENTION

After mating, behavioral and physiological changes are seen in thefemale insect. Compared to virgins, mated Drosophila melanogasterfemales are largely unreceptive to further mating, lay eggs at anelevated rate, live less long, and store and efficiently utilize sperm(reviewed in Hall, 1994; Chen, 1996; Kubli, 1996; Wolfner, 1997). Thesechanges in the female occur because she receives, via seminal fluid,secretions from her mate's accessory gland and also sperm (see Chen,1996; Kubli, 1996; Wolfner, 1997 for reviews and for originalreferences). Products of the predominant cell type of the accessorygland, the main cells, are necessary for changes in the female'segg-laying rate and receptivity on the first day after mating. Storedsperm cause these effects to persist for up to 11 days following mating.Accessory gland main cell secretions also shorten the life span of themated female. In addition, they play a role in the storage of sperm andin the competition between sperm from sequential matings.

Knowledge of how accessory gland products mediate these changes isimportant in understanding the control of insect fertility and themechanisms of peptide hormone action. Once genes encoding Accessorygland proteins (Acps) are identified, genetic and molecular genetictechniques uniquely possible in Drosophila can be used to dissect therole of each protein in reproduction. In a few cases, it is possible toidentify the functions of Acps by injecting purified fractions intounmated female flies and observing behavioral effects. For example, inD. melanogaster, a “sex peptide” (“SP”) of 36 amino acids was purifiedand shown to stimulate egg-laying and depress receptivity to mating forone day (Chen, 1988). SP was cloned and shown to derive from a singlegene at chromosomal position 70A (Chen, 1988). A sex peptide and asecond peptide, ovulation-stimulating substance (OSS), with similaractivities have also been purified from D. suzukii (Ohashi, 1991;Schmidt, 1993).

Only Acps which can be purified or synthesized in active forms, act ontheir own and act via the hemolymph, can be identified by such assays.In order to identify Acp genes without presupposition of function,differential cDNA hybridization can be used to isolate RNAs expressedonly in accessory glands (Schäfer, 1986; DiBenedetto, 1987; Monsma andWolfner, 1988). cDNA hybridization screens are more likely to isolateabundant RNAs in a tissue. Thus, they are biased towards RNAs expressedin main cells of the accessory gland (96% of the secretory cells of theaccessory gland; Bertram, 1992) rather than the rarer secondary cells(4% of the secretory cells of the gland; Bertram, 1992). Previousdifferential cDNA hybridization screens for genomic clones encodingmale-specific transcripts identified three genomic regions encoding Acps(Schäfer, 1986; DiBenedetto, 1987). Of these, the 95EF region encodes asmall secreted Acp (DiBenedetto, 1990), 57D contains a gene clusterencoding three small peptides (Simmerl, 1995), and the 51F locus has notyet been characterized. In addition to these genes, a region encodingtwo Acps has been identified by screening a “chromosomal walk” foraccessory gland-specific transcription units (Monsma and Wolfner, 1988).In this region, only 20 bases separate the gene for Acp26Aa, anELH-similar prohormone-like molecule (Monsma and Wolfner, 1988) thatstimulates egg-laying in the mated female fly (Herndon and Wolfner,1995), from the gene for Acp26Ab, a small peptide of as yet unknownfunction (Monsma and Wolfner, 1988). The previously-isolated Acp genesare only a small subset of Acp genes, though the total number of Acpgenes is difficult to estimate from prior protein electrophoretic data(e.g. Ingman-Baker and Candido, 1980; Stumm-Zollinger and Chen, 1985;Whalen and Wilson, 1986; Coulthart and Singh, 1988) as summarized anddiscussed in Chen (1991). This is because on the one hand in theelectrophoretic studies small peptides were not resolved, while on theother hand some Acps run as multiple bands on SDS gels (Monsma andWolfner, 1988). To gain a more complete picture of the spectrum ofproteins produced by the accessory gland, a differential screen aimeddirectly at accessory gland-specific RNAs was performed.

SUMMARY OF THE INVENTION

The present invention provides an isolated nucleic acid moleculeencoding an accessory gland protein from Drosophila which has thebiological property of an insect toxin.

The invention further provides an isolated nucleic acid moleculeencoding an amino acid sequence sufficiently duplicative of theaccessory gland protein encoded by the nucleic acid molecule of SEQ IDNO:2 so that a polypeptide expressed from the nucleic acid molecule hasthe biological property of an insect toxin.

Another embodiment of the invention is an isolated Drosophilamelanogaster insect toxin protein.

The invention also provides a method of reducing an insect's life span.The insect is contacted with an isolated Drosophila insect toxin proteinunder conditions effective shorten the insect's life span.

Yet another embodiment of the invention is a method of reducing aninsect's life span by contacting the insect with an expression vectorcontaining a nucleic acid molecule encoding a Drosophila insect toxinprotein under conditions effective to express the protein.

The invention also provides isolated nucleic acid molecules having thenucleotide sequences of SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ IDNO:13, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ IDNO:30, or a nucleic acid molecules which hybridizes under stringentconditions to a nucleic acid molecule having a nucleic acid sequence ofSEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:18, SEQID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:30.

The present invention also provides isolated proteins having an aminoacid sequence of SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO: 12,SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ ID NO:26,SEQ ID NO:29, or SEQ ID NO:32.

Yet another embodiment of the invention is a method for determiningwhether a female Drosophila melanogaster has recently mated. Anantibody, fragment thereof, or probe which recognizes a protein having asequence provided in SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ IDNO:26, SEQ ID NO:29, or SEQ ID NO:32 is provided. The antibody, fragmentthereof, or probe is bound to a label effective to permit detection ofthe protein upon binding of the antibody, fragment thereof, or probe tothe protein. The labeled antibody is contacted with a fluid or tissuesample from Drosophila melanogaster under conditions effective to permitbinding of the antibody, fragment thereof, or probe to the protein. Thepresence of any of the protein in the biological sample is detected bydetecting the label.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the genomic organization and transcript characteristicsof new Acp genes. Gene name, position and copy number: The genes arenamed Acp followed by their location on the polytene chromosomes asdetermined by in situ hybridization. The site for Acp76A is determinedas 75F/76A, for 63F is 63F/64A, for 31F is 31F/32A. The genomic clonecontaining Acp33A has a minor site of hybridization at 32E. Restrictionmap: The map of the genomic region containing the accessory glandtranscript is shown, to a scale such that the leftmost EcoRI-XbaIfragment of Acp31F is 1.0 kb. In all cases but 2, the map is derivedfrom a single genomic clone of the region. The larger region covered forAcps 36DE and 33A is a composite of the overlapping maps of threegenomic clones each. The genomic fragment(s) that hybridize to the Acptranscript is shown as a thicker line. The clones are oriented with theAcp gene transcription direction from left to right, except for Acps31F, 53Eb and rep1, where the transcription direction was notdetermined. E=Eco RI, H=Hin dIII, B=Bam HI, X=Xba I, S=Sal I, Bgl=BglII, N=Ngo M1, P=Pml I, W=Swa I. Transcript characteristics: The size ofeach poly A⁺ accessory gland transcript was determined on a minimum ofthree independent Northern blots. The number of bases (nt) in the cDNAclone before the first AUG is listed (5′ UTR); since the cDNA clones maynot be full-length, the size of this untranslated leader should be takenas a minimum. For all genes except Acp36DE, the longest open readingframe begins with the first AUG. As noted in the text, although Acp36DEhas a short open reading frame beginning at base 18 of the cDNA sequence(and preceded by AUUA), the Acp is encoded by the longer second openreading frame which is the one listed in the figure.

The four bases immediately 5′ of the initiating AUG are shown; thosematching consensus (C/A A A C/A; Cavener, 1987) are underlined. Thecolumn headed 3′UTR gives the number of bases from the translationalstop codon to the beginning of the poly A tail. For Acp53Ea, the cDNAclone did not contain the polyA tail. Thus the size of its UTR is aminimum. The distance of that tail from the AAUAAA signal (or its best,5/6, match) is given in the “pA sig.” column.

The GenBank accession numbers for these sequences are given. For Acps32CD and 98AB, the sequences reported are composites of overlappinggenomic and cDNA sequences. GenBank requires that genomic and cDNAsequences be given separate accession numbers. For each of these genes,the first number listed is for the genomic sequence, the second for itspartially-overlapping cDNA. For Acp76A, the accession number is forgenomic sequence, which completely contains the cDNA sequence of thisapparently intron-less mRNA. The other accession numbers are for cDNAsequences. Two accession numbers are given for Acp33A, since GenBankrequires that each predicted ORF in this single mRNA be listed with itsown accession number. The nucleotide sequences for these two accessionnumbers are, of course, the same. Characteristics of predicted Acp: Thenumber of amino acids (aa) in the Acp-encoding ORF, the length of thehydrophobic sequence terminating at a predicted signal sequence cleavagesite (von Heijne, 1983) and predicted features of the Acp are listed insuccessive columns.

FIGS. 2A and 2B show the expression of an accessory gland gene, Acp36DE,as an example. FIG. 2A is a representative Northern blot of maleaccessory gland RNA and female RNA probed with radiolabeled RNAcomplementary to Acp36DE. A male specific RNA of 2.6-2.7 kb is detected.The blot was probed separately with sequences complementary to actin asa loading control as in DiBenedetto (1987), which confirmed thatequivalent amounts of RNA were present in the two lanes. FIG. 2Bprovides a representative in situ hybridization to whole-mount accessoryglands from a 3-day-old virgin male. The probe was thedigoxigenin-labeled 2.1 kb EcoRI fragment of Acp36DE cDNA clone #11A(Bertram, 1994). Staining is seen in the accessory glands (“ag”), butnot in the ejaculatory duct (“ed”). Bar=0.128 millimeter. FIG. 2B(inset) is a higher magnification of the distal tip of an accessorygland from a similar in situ hybridization. Main cells are stained.Secondary cells are not stained. This can be seen most clearly at theedge of the gland, where an arrowhead points to an unstained secondarycell (the apparent “bite” taken out of the edge of the stained tissue isthe unstained cell). Other secondary cells, lying atop a layer ofstained main cells and themselves surrounded by stained main cells, areseen as light circles. Bar=0.051 millimeter.

FIG. 3 provides the predicted protein sequences of new Acps. Thepredicted protein sequence from the single long ORF in each Acp's cDNAis shown. For Acp33A, both ORFs are shown; ORF1 is the more 5′ one. Thepotential signal sequence is written in lower case. Potential N-linkedglycosylation sites (N-x-S/T/C; Kornfeld and Kornfeld, 1985; Miletichand Broze, 1990), amidation site (IGKK; Kreil, 1984; Bradbury and Smyth,1987), glycosaminoglycan attachment sites (SGxG; Hassell, 1986; Bourdon,1987) and basic amino acids (K, R) in contexts consistent withprohormone processing cleavages (Schwartz, 1986; Benoit, 1987; Nakayama,1992) are underlined. The amino acids that match serpin consensus inAcp76A, and those in Acp62F that are similar to toxin PhTx2-6 are boxed.Sequencing of genomic DNA upstream from the 5′ end of the incompletecDNA of Acp32CD led to the discovery of an ORF encoding 329 amino acids.This ORF is too long to be encoded on the 0.95 kb Acp32CD RNA, whosesize includes a poly A tail. Therefore, it is surmised that Acp32CDcorresponds to the 241 amino acid sequence shown in the figure, whichbegins at the second AUG of the 329-amino acid ORF (AGAU immediatelyprecedes the AUG of the long ORF). The amino acids encoded by genomicsequence immediately upstream of the sequence shown in this figure are:mppllrhcfg hafiglplfn GQEQPRPQSN RFDSGQRRSS LYIRDGRTAR AAQRCSDVADADAATHWLLG PVALGQLPEH GALGQKYY (SEQ ID NO:1).

FIG. 4 is a best-fit alignment of similar regions of Acp62F (aa residues46-73 of SEQ ID NO:4) and PhTx2-6 (SEQ ID NO:35, aa residues 9-35 of thefull length PhTx2-6 protein).

FIG. 5 shows the rate of survival of baculovirus injected 5^(th) instarTrichoplusia ni. The results of controls and baculovirus which expressAcp62F are included.

FIG. 6 shows the rate of pupation of 3^(rd) instar Trichoplusia ni afterinjection with Acp62F protein.

FIG. 7 shows the effect on survival of Acp62F expression in pre-adultfruit flies.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides isolated genes encoding 12 previouslyunreported accessory gland-specific mRNAs from the fruit fly Drosophilamelanogaster. The restriction maps of the new genes, the chromosomepositions—which are all autosomal—of the 11 non-repetitive genes, theirexpression patterns, and the sequences of the Accessory gland proteins(Acps) encoded by 9 of the genes are provided.

The present invention provides an isolated nucleic acid moleculeencoding an accessory gland protein from Drosophila which has thebiological property of an insect toxin. The 115 amino acid Acp62F has a28 amino acid region of high sequence similarity to a neurotoxin of theBrazilian armed spider Phoneutria nigriventer. In a preferred embodimentof the invention, the accessory gland protein has greater than 40%sequence similarity to amino acids 9 to 35 of the PhTx2-6 toxin ofPhoneutria nigriventer. This protein has been tested and has been shownto kill or inhibit the growth of insects. In particular, the accessorygland protein is toxic to Drosophila or trichoplusia.

The present invention provides a gene encoding Acp62F. The cDNA sequenceof the Acp62F gene is as follows (SEQ ID NO:2):

1 ggtagacgta ttccccatct acaatgacgg acatgtggag cttgaagatc tgtgcctgtc 61tgggccttct attacttttc aaacccatcg actccatggg atggcaagga cctaaagttg 121actgtacggc caacggaact cagacggagt gtcctgtagc atgtcctgaa acctgcgagt 181actccggcaa tggaccctgc gtcaagatgt gcggagctcc ttgtgtgtgt aagccgggat 241atgttatcaa tgagaggatt ccggcctgtg ttctgcgatc cgattgccca aaagatgttg 301ttcgaaagga agatatgcta ctgggtgtat cgaactttaa gtgctttagc agaaattaca 361actgttcata gaaatttatt aggaaggcag ctaaacttta aactaaaata caataaaatg 421taaataaaaa aaaaaaaaa

In a preferred embodiment of the invention, the isolated nucleic acidmoleucle has a nucleotide sequence according to SEQ ID NO:2, or anucleic acid molecule which hybridizes under stringent conditions to anucleic acid molecule having the nucleic acid sequence of SEQ ID NO:2.

The nucleic acid molecule can be deoxyribonucleic acid (DNA) orribonucleic acid (RNA), genomic or recombinant, biologically isolated orsynthetic. The invention encompasses the DNA sequences as well as theircomplements. The DNA molecule can be a cDNA molecule, which is a DNAcopy of a messenger RNA (mRNA) encoding the accessory gland protein. Asuitable RNA molecule is mRNA.

Suitable nucleic acid molecules include those nucleic acid moleculesencoding an accessory gland protein and having a nucleotide sequencewhich is at least 95% homologous to the nucleotide sequence of anaccessory gland protein of the present invention.

While the nucleotide sequence is at least 95% homologous, nucleotideidentity is not required. As should be readily apparent to those skilledin the art, various nucleotide substitutions are possible which aresilent mutations (i.e. the amino acid encoded by the particular codondoes not change). It is also possible to substitute a nucleotide whichalters the amino acid encoded by a particular codon, where the aminoacid substituted is a conservative substitution (i.e. amino acid“homology” is conserved).

Alternatively, suitable DNA sequences may be identified by hybridizationto the disclosed sequences which encode the accessory gland proteinsunder stringent conditions. For example, sequences can be isolated thathybridize to a DNA molecule comprising a nucleotide sequence ofapproximately 50 continuous bases of the sequences encoding theaccessory gland proteins under stringent conditions characterized by ahybridization buffer comprising 0.9M sodium citrate (“SSC”) buffer at atemperature of 37° C. and remaining bound when subject to washing withthe SSC buffer at 37° C.; and preferably in a hybridization buffercomprising 20% formamide in 0.9M saline/0.09M SSC buffer at atemperature of 42° C. and remaining bound when subject to washing at 42°C. with 0.2×SSC buffer at 42° C.

Another embodiment of the invention is an expression vector carrryingthe nucleic acid molecule encoding Acp62F.

The DNA molecule encoding the accessory gland proteins can beincorporated in cells using conventional recombinant DNA technology.Generally, this involves inserting the DNA molecule into an expressionsystem to which the DNA molecule is heterologous (i.e. not normallypresent). The heterologous DNA molecule is inserted into the expressionsystem or vector in proper sense orientation and correct reading frame.The vector contains the necessary elements for the transcription andtranslation of the inserted protein-coding sequences.

U.S. Pat. No. 4,237,224 to Cohen and Boyer, which is hereby incorporatedby reference, describes the production of expression systems in the formof recombinant plasmids using restriction enzyme cleavage and ligationwith DNA ligase. These recombinant plasmids are then introduced by meansof transformation and replicated in unicellular cultures includingprocaryotic organisms and eucaryotic cells grown in tissue culture.

Recombinant genes may also be introduced into viruses, such as vaccinavirus. Recombinant viruses can be generated by transfection of plasmidsinto cells infected with virus.

Suitable vectors include, but are not limited to, the following viralvectors such as lambda vector system gt11, gt WES.tB, Charon 4, andplasmid vectors such as pBR322, pBR325, pACYC177, pACYC1084, pUC8, pUC9,pUC18, pUC19, pLG339, pR290, pKC37, pKC101, SV 40, pBluescript II SK +/−or KS +/− (see Stratagene, 1993), pQE, pIH821, pGEX, pET series (seeStudier, 1990), and any derivatives thereof. Recombinant molecules canbe introduced into cells via transformation, particularly transduction,conjugation, mobilization, or electroporation. The DNA sequences arecloned into the vector using standard cloning procedures in the art, asdescribed by Sambrook, (1989).

A variety of host-vector systems may be utilized to express theprotein-encoding sequence(s). Primarily, the vector system must becompatible with the host cell used. Host-vector systems include but arenot limited to the following: bacteria transformed with bacteriophageDNA, plasmid DNA, or cosmid DNA; microorganisms such as yeast containingyeast vectors; mammalian cell systems infected with virus (e.g.,vaccinia virus, adenovirus, etc.); insect cell systems infected withvirus (e.g., baculovirus); and plant cells infected by bacteria. Theexpression elements of these vectors vary in their strength andspecificities. Depending upon the host-vector system utilized, any oneof a number of suitable transcription and translation elements can beused.

In a preferred embodiment, the expression vector is a viral vector. In amore preferred embodiment, the viral vector is a baculovirus vector.Baculovirus vectors, which are derived from the genome of the AcNPVvirus, are designed to provide high level expression of cDNA in the Sf9line of insect cells. A baculovirus vector is constructed in accordancewith techniques that are known in the art, for example, as described inKitts, (1993); Smith, (1983); and Luckow and Summer, (1989). In oneembodiment of the present invention, a baculovirus expression vector isconstructed substantially in accordance to Summers and Smith, (1987).Moreover, materials and methods for baculovirus/insect cell expressionsystems are commercially available in kit form, for example, the MaxBacRegistered TM kit from Invitrogen (San Diego, Calif.). Recombinantbaculoviruses are generated by homologous recombination followingco-transfection of the baculovirus transfer vector and linearized AcNPVgenomic DNA (Kitts, 1990) into Sf9 cells.

In yet another embodiment, the invention provides a host cell having theexpression vector carrying the accessory gland protein gene. In apreferred embodiment, the host cell is an insect cell.

Different genetic signals and processing events control many levels ofgene expression (e.g., DNA transcription and messenger RNA (mRNA)translation).

Transcription of DNA is dependent upon the presence of a promoter whichis a DNA sequence that directs the binding of RNA polymerase and therebypromotes mRNA synthesis. The DNA sequences of eucaryotic promotersdiffer from those of procaryotic promoters. Furthermore, eucaryoticpromoters and accompanying genetic signals may not be recognized in ormay not function in a procaryotic system, and, further, procaryoticpromoters are not recognized and do not function in eucaryotic cells.

Similarly, translation of mRNA in procaryotes depends upon the presenceof the proper procaryotic signals which differ from those of eucaryotes.Efficient translation of mRNA in procaryotes requires a ribosome bindingsite called the Shine-Dalgarno (“SD”) sequence on the mRNA. Thissequence is a short nucleotide sequence of mRNA that is located beforethe start codon, usually AUG, which encodes the amino-terminalmethionine of the protein. The SD sequences are complementary to the3′-end of the 16S rRNA (ribosomal RNA) and probably promote binding ofmRNA to ribosomes by duplexing with the rRNA to allow correctpositioning of the ribosome. For a review on maximizing gene expression,see Roberts and Lauer, (1979).

Promoters vary in their “strength” (i.e. their ability to promotetranscription). For the purposes of expressing a cloned gene, it isdesirable to use strong promoters in order to obtain a high level oftranscription and, hence, expression of the gene. Depending upon thehost cell system utilized, any one of a number of suitable promoters maybe used. For instance, when cloning in E. coli, its bacteriophages, orplasmids, promoters such as the T7 phage promoter, lac promoter, trppromoter, recA promoter, ribosomal RNA promoter, the PR and PL promotersof coliphage lambda and others, including but not limited, to lacUV5,ompF, bla, lpp, and the like, may be used to direct high levels oftranscription of adjacent DNA segments. Additionally, a hybridtrp-lacUV5 (tac) promoter or other E. coli promoters produced byrecombinant DNA or other synthetic DNA techniques may be used to providefor transcription of the inserted gene.

Bacterial host cell strains and expression vectors may be chosen whichinhibit the action of the promoter unless specifically induced. Incertain operations, the addition of specific inducers is necessary forefficient transcription of the inserted DNA. For example, the lac operonis induced by the addition of lactose or IPTG(isopropylthio-beta-D-galactoside). A variety of other operons, such astrp, pro, etc., are under different controls.

Specific initiation signals are also required for efficient genetranscription and translation in procaryotic cells. These transcriptionand translation initiation signals may vary in “strength” as measured bythe quantity of gene specific messenger RNA and protein synthesized,respectively. The DNA expression vector, which contains a promoter, mayalso contain any combination of various “strong” transcription and/ortranslation initiation signals. For instance, efficient translation inE. coli requires an SD sequence about 7-9 bases 5′ to the initiationcodon (“ATG”) to provide a ribosome binding site. Thus, any SD-ATGcombination that can be utilized by host cell ribosomes may be employed.Such combinations include but are not limited to the SD-ATG combinationfrom the cro gene or the N gene of coliphage lambda, or from the E. colitryptophan E, D, C, B or A genes. Additionally, any SD-ATG combinationproduced by recombinant DNA or other techniques involving incorporationof synthetic nucleotides may be used.

Once the isolated DNA molecule encoding the accessory gland protein hasbeen cloned into an expression system, it is ready to be incorporatedinto a host cell. Such incorporation can be carried out by the variousforms of transformation noted above, depending upon the vector/host cellsystem. Suitable host cells include, but are not limited to, bacteria,virus, yeast, mammalian cells, insect, plant, and the like.

The invention also provides an antisense nucleic acid molecule that iscomplementary to the mRNA encoding the accessory gland protein, or afragment thereof capable of hybridizing under stringent conditions tothe mRNA. The antisense nucleic acid molecule is ribonucleic acid. Thisantisense molecule can base-pair with the mRNA, preventing translationof the mRNA into protein.

The invention further provides an isolated fragment of the nucleic acidmolecule encoding the accessory gland protein. Nucleic acid moleculesencoding the accessory gland proteins, and fragments of the nucleic acidmolecules, are thus provided.

Each of the nucleic acid molecules, fragments thereof, antisense nucleicacid molecules, and fragments thereof, can be expressed in suitable hostcells using conventional techniques. Such techniques may involve the useof expression vectors which comprise the nucleic acid molecules,fragments thereof, antisense nucleic acid molecules, or fragmentsthereof. These expression vectors can then be used to transform suitablehost cells.

Host cells transformed with nucleic acid molecules encoding theaccessory gland protein can be used to produce accessory gland proteins(or cells transformed with the fragments can be used to producefragments of the accessory gland proteins). Alternatively, the fragmentsor full-length accessory gland proteins can be produced syntheticallyusing the sequence information of the accessory gland proteins andfragments. In host cells transformed with the antisense nucleic acidmolecules, or fragments thereof, the antisense nucleic acid molecules orfragments thereof will block translation of the accessory gland protein.Accordingly, in host cells transformed with the antisense nucleic acidmolecules or fragments thereof, the expression of accessory glandprotein is decreased.

The invention also provides an isolated nucleic acid molecule having anucleic acid sequence encoding an amino acid sequence sufficientlyduplicative of the accessory gland protein encoded by the disclosednucleic acid molecules so that a polypeptide expressed from the nucleicacid molecule has the biological property of an insect toxin. The cDNAfor Acp62F includes an open reading frame beginning at position 24 ofthe cDNA sequence. The open reading frame sequence is provided in SEQ IDNO:3, as follows:

1 atgacggaca tgtggagctt gaagatctgt gcctgtctgg gccttctatt acttttcaaa 61cccatcgact ccatgggatg gcaaggacct aaagttgact gtacggccaa cggaactcag 121acggagtgtc ctgtagcatg tcctgaaacc tgcgagtact ccggcaatgg accctgcgtc 181aagatgtgcg gagctccttg tgtgtgtaag ccgggatatg ttatcaatga gaggattccg 241gcctgtgttc tgcgatccga ttgcccaaaa gatgttgttc gaaaggaaga tatgctactg 301ggtgtatcga actttaagtg ctttagcaga aattacaact gttca

The present invention also provides an isolated Drosophila melanogasterinsect toxin protein. The amino acid sequence of the Acp62F protein isprovided in SEQ ID NO:4, as follows:

1 MTDMWSLKIC ACLGLLLLFK PIDSMGWQGP KVDCTANGTQ TECPVACPET CEYSGNGPCV 61KMCGAPCVCK PGYVINERIP ACVLRSDCPK DVVRKEDMLL GVSNFKCFSR NYNCS

In a preferred embodiment, the isolated Drosophila melanogaster insecttoxin protein has greater than 40% sequence similarity to amino acids 9to 35 of the PhTx2-6 toxin of Phoneutria nigriventer. Another preferredembodiment is where the isolated Drosophila melanogaster insect toxinprotein is toxic to Drosophila or caterpillar.

The isolated Drosophila melanogaster insect toxin protein may have anamino acid sequence according to SEQ ID NO:4, or be a protein encoded bya nucleic acid molecule which hybridizes under stringent conditions to anucleic acid molecule having the nucleic acid sequence of SEQ ID NO:2and which has the biological property of an insect toxin.

The protein or polypeptide of the present invention is preferablyproduced in purified form (preferably at least about 80%, morepreferably 90%, pure) by conventional techniques. In accordance with thepresent invention, the accessory gland proteins are isolated from hostcells. Methods for protein isolation are known in the art. Generally,proteins can be purified by conventional chromatography, includinggel-filtration, ion-exchange, and immunoaffinity chromatography, byhigh-performance liquid chromatography, such as reversed-phasehigh-performance liquid chromatography, ion-exchange high-performanceliquid chromatography, size-exclusion high-performance liquidchromatography, high-performance chromatofocusing and hydrophobicinteraction chromatography, etc., by electrophoretic separation, such asone-dimensional gel electrophoresis, two-dimensional gelelectrophoresis, etc. Such methods are known in the art. See for exampleAusubel (1994). Additionally, antibodies can be prepared againstsubstantially pure preparations of the protein. See, for example, Radka,(1983) and Radka, (1984). Any combination of methods may be utilized topurify protein having pesticidal properties. As the protocol is beingformulated, pesticidal activity is determined after each purificationstep, if relevant.

Fragments of the above polypeptide or protein are also encompassed bythe present invention. Suitable fragments can be produced by severalmeans. In the first, subclones of the gene encoding the protein of thepresent invention are produced by conventional molecular geneticmanipulation by subcloning gene fragments. The subclones then areexpressed in vitro or in vivo in bacterial cells to yield a smallerprotein or peptide that can be tested for an accessory gland protein.

As an alternative, fragments of an accessory gland protein can beproduced by digestion of an accessory gland protein with proteolyticenzymes like chymotrypsin or Staphylococcus proteinase A, or trypsin.Different proteolytic enzymes are likely to cleave accessory glandproteins at different sites based on the amino acid sequence of anaccessory gland protein.

In another approach, based on knowledge of the primary structure of theprotein, fragments of the accessory gland protein gene may besynthesized by using the PCR technique together with specific sets ofprimers chosen to represent particular portions of the protein. Thesethen would be cloned into an appropriate vector for increased expressionof an accessory peptide or protein.

Chemical synthesis can also be used to make suitable fragments. Such asynthesis is carried out using known amino acid sequences for theaccessory gland protein being produced. Alternatively, subjecting a fulllength accessory gland protein to high temperatures and pressures willproduce fragments. These fragments can then be separated by conventionalprocedures (e.g., chromatography, SDS-PAGE).

Variants may also (or alternatively) be modified by, for example, thedeletion or addition of amino acids that have minimal influence on theproperties, secondary structure, and hydropathic nature of thepolypeptide. For example, a polypeptide may be conjugated to a signal(or leader) sequence at the N-terminal end of the protein whichco-translationally or post-translationally directs transfer of theprotein. The polypeptide may also be conjugated to a linker or othersequence for ease of synthesis, purification, or identification of thepolypeptide.

Gene amplification can also be used to obtain very high levels ofexpression of transfected gene. When cell cultures are treated withmethotrexate (“Mtx”), an inhibitor of a critical metabolic enzyme,dihydrofolate reductase (“DHFR”), most cells die, but eventually someMtx-resistant cells grow up. A gene to be expressed in cells iscotransfected with a cloned DHFR gene, and the transfected cells aresubjected to selection with a low concentration of Mtx. Resistant cellsthat have taken up the DHFR gene (and, in most cases, the cotransfectedgene) multiply. Increasing the concentration of Mtx in the growth mediumin small steps generates populations of cells that have progressivelyamplified the DHFR gene, together with linked DNA. Although this processtakes several months, the resulting cell cultures capable of growing inthe highest Mtx concentrations will have stably amplified the DNAencompassing the DHFR gene a hundredfold or more, leading to significantelevation of the expression of the cotransfected gene.

Once the nucleic acid molecule encoding an accessory gland protein hasbeen inserted into a host cell, with or without the use of anintermediate expression vector, the host cell can be used to produce theaccessory gland protein by culturing the cell under conditions suitablefor translation of the DNA molecule, thereby expressing the accessorygland protein. The accessory gland protein can then be recovered fromthe cell. Generally, the accessory gland protein of the presentinvention is produced in purified form by conventional techniques, suchas by secretion into the growth medium of recombinant E. coli. Toisolate the protein, the E. coli host cell carrying a recombinantplasmid is propagated, homogenized, and the homogenate is centrifuged toremove bacterial debris. The supernatant is then subjected to sequentialammonium sulfate precipitation. The fraction containing the protein ofthe present invention is subjected to gel filtration in an appropriatelysized dextran or polyacrylamide column to separate the proteins. Ifnecessary, the protein fraction may be further purified by HPLC.

The present invention also provides an isolated antibody, fragmentthereof, or probe which recognizes an accessory gland protein providedby the invention. Preferred antibodies, fragments thereof, or probes arethose which recognize a protein having an amino acid sequence accordingto SEQ ID NO:4, or a protein encoded by a nucleic acid molecule whichhybridizes under stringent conditions to a nucleic acid molecule havingthe nucleic acid sequence of SEQ ID NO:2 and where the protein has thebiological function of an insect toxin.

Antibodies can also be raised to each of the accessory gland proteins,and to the isolated fragments thereof. Antibodies of the subjectinvention include polyclonal antibodies and monoclonal antibodies whichare specific for an accessory gland protein or isolated fragmentsthereof. In addition to utilizing whole antibodies, the presentinvention encompasses use of binding portions of such antibodies. Suchbinding portions include Fab fragments, F(ab′)2 fragments, and Fvfragments. Such antibody fragments can be made by conventionalprocedures, such as proteolytic fragmentation procedures, as describedin Goding, (1983). These antibodies or fragments thereof can thus beused to detect the presence of an accessory gland protein in a sample(or to detect the presence of a fragment of an accessory gland protein),by contacting the sample with the antibody or fragment thereof. Theantibody or fragment thereof binds to an accessory gland protein orfragment thereof present in the sample, forming a complex therewith. Thecomplex can then be detected, thereby detecting the presence of theaccessory gland protein or fragment thereof in the sample.

In a preferred embodiment the antibody, fragment thereof, or probe is amonoclonal antibody. Alternatively, the antibody, fragment thereof, orprobe is a polyclonal antibody.

Monoclonal antibody production may be effected by techniques which arewell-known in the art. Basically, the process involves first obtainingimmune cells (lymphocytes) from the spleen of a mammal (e.g., mouse)which has been previously immunized with the antigen of interest eitherin vivo or in vitro. The antibody-secreting lymphocytes are then fusedwith (mouse) myeloma cells or transformed cells, which are capable ofreplicating indefinitely in cell culture, thereby producing an immortal,immunoglobulin-secreting cell line. The resulting fused cells, orhybridomas, are cultured, and the resulting colonies screened for theproduction of the desired monoclonal antibodies. Colonies producing suchantibodies are cloned and grown either in vivo or in vitro to producelarge quantities of antibody. A description of the theoretical basis andpractical methodology of fusing such cells is set forth in Kohler andMilstein, (1975).

Mammalian lymphocytes are immunized by in vivo immunization of theanimal (e.g., a mouse) with the protein or polypeptide of the presentinvention. Such immunizations are repeated as necessary at intervals ofup to several weeks to obtain a sufficient titer of antibodies.Following the last antigen boost, the animals are sacrificed and spleencells removed.

Fusion with mammalian myeloma cells or other fusion partners capable ofreplicating indefinitely in cell culture is effected by standard andwell-known techniques, for example, by using polyethylene glycol (“PEG”)or other fusing agents (See Milstein and Kohler, (1976). This immortalcell line, which is preferably murine, but may also be derived fromcells of other mammalian species, including but not limited to rats andhumans, is selected to be deficient in enzymes necessary for theutilization of certain nutrients, to be capable of rapid growth, and tohave good fusion capability. Many such cell lines are known to thoseskilled in the art, and others are regularly described.

Procedures for raising polyclonal antibodies are also well known.Typically, such antibodies can be raised by administering the protein orpolypeptide of the present invention subcutaneously to New Zealand whiterabbits which have first been bled to obtain pre-immune serum. Theantigens can be injected at a total volume of 100 ml per site at sixdifferent sites. Each injected material will contain adjuvants with orwithout pulverized acrylamide gel containing the protein or polypeptideafter SDS-polyacrylamide gel electrophoresis. The rabbits are then bledtwo weeks after the first injection and periodically boosted with thesame antigen three times every six weeks. A sample of serum is thencollected 10 days after each boost. Polyclonal antibodies are thenrecovered from the serum by affinity chromatography using thecorresponding antigen to capture the antibody. This and other proceduresfor raising polyclonal antibodies are disclosed in Harlow, (1988).

In addition to utilizing whole antibodies, the processes of the presentinvention encompass use of binding portions of such antibodies. Suchbinding portions include Fab fragments, F(ab′)2 fragments, and Fvfragments. These antibody fragments can be made by conventionalprocedures, such as proteolytic fragmentation procedures, as describedin Goding, (1983).

The present invention also provides a method of reducing an insect'slife span. The insect is contacted with an isolated Drosophila insecttoxin protein under conditions effective shorten the insect's life span.In a preferred embodiment, the isolated Drosophila insect toxin proteinused to shorten the life span of the insect has an amino acid sequencecorresponding to SEQ ID NO:4.

The active ingredients of the present invention are normally applied inthe form of compositions and can be applied to the crop area or plant tobe treated, simultaneously or in succession, with other compounds. Thesecompounds can be both fertilizers or micronutrient donors or otherpreparations that influence plant growth. They can also be selectiveherbicides, insecticides, fungicides, bactericides, nematicides,mollusicides or mixtures of several of these preparations, if desired,together with further agriculturally acceptable carriers, surfactants orapplication-promoting adjuvants customarily employed in the art offormulation. Suitable carriers and adjuvants can be solid or liquid andcorrespond to the substances ordinarily employed in formulationtechnology, e.g. natural or regenerated mineral substances, solvents,dispersants, wetting agents, tackifiers, binders or fertilizers.Preferred methods of applying an active ingredient of the presentinvention or an agrochemical composition of the present invention whichcontains at least one of the pesticidal proteins produced by thebacterial strains of the present invention are leaf application, seedcoating and soil application. The number of applications and the rate ofapplication depend on the intensity of infestation by the correspondingpest.

In an alternative embodiment of the invention, the gene encoding theinsect toxin protein may be introduced into plants. The gene is underthe control of a promoter which is effective in plants, so that theinsect toxin gene is expressed in the plant. The insects would ingestthe protein when eating any portion of the plant. When utilizingtransgenic plants, this involves providing a transgenic planttransformed with a DNA molecule encoding a fragment of a insect toxinprotein, which fragment is toxic to the target insect, and growing theplant under conditions effective to permit that DNA molecule to controlinsects. Alternatively, a transgenic plant seed transformed with a DNAmolecule encoding a fragment of a hypersensitive response elicitorpolypeptide or protein which fragment is toxic to the target insects canbe provided and planted in soil. A plant is then propagated from theplanted seed under conditions effective to permit that DNA molecule tocontrol insects.

In addition to contacting the insect with the protein of the invention,the invention provides a method of reducing an insect's life span bycontacting the insect with an expression vector containing a nucleicacid molecule encoding a Drosophila insect toxin protein underconditions effective to express the protein. In a preferred embodiment,the Drosophila insect toxin protein is a protein having an amino acidsequence according to SEQ ID NO:4, or a protein encoded by a nucleicacid molecule which hybridizes under stringent conditions to a nucleicacid molecule having the nucleic acid sequence of SEQ ID NO:2 and wherethe protein has the biological function of an insect toxin.

The present invention also provides several other accessory glandproteins and the genes encoding the proteins. As discussed above, thegenes are named Acp followed by their location on the polytenechromosomes as determined by in situ hybridization. For example, thesite for Acp76A is determined as 75F/76A, for 63F is 63F/64A, for 31F is31F/32A. The genomic clone containing Acp33A has a minor site ofhybridization at 32E. Eight of the proteins predicted from thesesequences begin with putative secretion signals. Following their signalsequences, three of the predicted molecules are peptides and the otherfive are larger polypeptides with characteristics of cleavableprohormones. The ninth molecule, which has an N-terminal hydrophobicregion but no consensus signal cleavage site, is predicted to be a 716amino acid glycoprotein. Of the 9 proteins, two have intriguingsimilarities to sequences in protein databases. Acp76A is a 388 aminoacid pro-protein which contains a signature sequence for the serpinclass of protease inhibitors.

The invention provides the isolated nucleic acid molecules which encodea number of novel accessory gland proteins. The preferred embodiments ofthe invention are nucleic acid molecules having a nucleotide sequence ofSEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:18, SEQID NO:21, SEQ ID NO:24, SEQ ID NO:27, or SEQ ID NO:30. The presentinvention also includes nucleic acid molecules which hybridizes understringent conditions to a nucleic acid molecule having a nucleic acidsequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:5,SEQ ID NO:8, SEQ ID NO:13, SEQ ID NO:18, SEQ ID NO:21, SEQ ID NO:24, SEQID NO:27, or SEQ ID NO:30. As above, the invention provides expressionvectors and host cells containing the nucleic acids of the invention.

In a preferred embodiment, the isolated nucleic acid molecule encoded aprotein having an amino acid sequence corresponding to SEQ ID NO:4, SEQID NO:7, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, SEQ IDNO:20, SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, or SEQ ID NO:32.

Preferred nucleic acid molecules are deoxyribonucleic acid molecules.

The present invention also provides isolated proteins encoded by theaccessory gland proteins. Preferred proteins have the amino acidsequences, or fragments thereof, provided in SEQ ID NO:4, SEQ ID NO:7,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:20,SEQ ID NO:23, SEQ ID NO:26, SEQ ID NO:29, or SEQ ID NO:32.

The present invention provides a gene identified as Acp29AB, its cDNAsequence is as follows (SEQ ID NO:5):

1 gacacgcttg aaatcttcca aactggacat gtacgcatct aacctcttat acctgttggc 61attatggaac ctttgggatc tcagtggtgg gcagcaggac attccgaacg gaaaggctac 121attgccaagt ccacaaacgc cgcaaaatac aatcgatcag attggtatta accagaatta 181ttggtttaca tacaacgcgc ttaaacaaaa cgaaacattg gcaattattg atacaatgga 241aatgcgcata gcaagtagct tgctggagtt taaggcccag atggaaatcc agcttcagcc 301gttaaagatt ataatgcgac accatgcatc caacatcaaa gcgtctaaca acatcaagat 361gagacgattc gagaaagttg gctccagaca ttttcacatc gagaagaatc taatgcaaac 421ttggtttgag gcatatgtca catgtcgtaa aatgaacggt catctggcga acatccagga 481tgagaaggag ctggatggca tcttggcgtt agcacccaac aatagctact ggatagatat 541atccaaactg gttgaaaatg gcggcacatt cgtctccacc ctaaccggac gagaaccctt 601ctttgttaaa tggaagagta atcaggatac aaaaaaaaag aatcaatgcg tttacatcta 661tgctaaagag atgtcctatg atgagtgttt tgaaaaaaaa tctttcgttt gccaagcaga 721ccagtgggcc taaacataaa gaaaatattg ttttgtagct tgaataataa aattataaaa 781aaaaaaaaaa aa

The cDNA encodes an open reading frame which is located at nucleotides29 to 733 of SEQ ID NO:5 (SEQ ID NO:6):

1 atgtacgcat ctaacctctt atacctgttg gcattatgga acctttggga tctcagtggt 61gggcagcagg acattccgaa cggaaaggct acattgccaa gtccacaaac gccgcaaaat 121acaatcgatc agattggtat taaccagaat tattggttta catacaacgc gcttaaacaa 181aacgaaacat tggcaattat tgaccagaat taaatgcgca tagcaagtag cttgccggag 241tttaaggccc agatggaaat ccagcttcag ccgttaaaga ttataatgcg acaccatgca 301tccaacatca aagcgtctaa caacatcaag atgagacgat tigagaaagt tggctccaga 361cattttcaca tcgagaagaa tctaatgcaa acttggtttg aggcatatgt cacatgtcgt 421aaaatgaacg gtcatctggc gaacatccag gatgagaagg agctggatgg catcttggcg 481ttagcaccca acaatagcta ctggatagat atatccaaac tggttgaaaa tggcggcaca 541ttcgtctcca ccctaaccgg acgagaaccc ttctttgtca aacggaagag taatcaggat 601acaaaaaaaa agaatcaatg cgtttacatc tatgctaaag agatgtccta tgatgagtgt 661tttgaaaaaa aatctttcgt ttgccaagca gaccagtggg cc

The amino acid sequence of the Acp29AB protein is provided as follows(SEQ ID NO:7):

1 MYASNLLYLL ALWNLWDLSG GQQDIPNGKA TLPSPQTPQN TIDQIGINQN YWFTYNALKQ 61NETLAIIDTM EMRIASSLLE FKAQMEIQLQ PLKIIMRHHA SNIKASNNIK MRRFEKVGSR 121HFHIEKNLMQ TWFEAYVTCR KMNGHLANIQ DEKELDGILA LAPNNSYWID ISKLVENGGT 181FVSTLTGREP FFVKWKSNQD TKKKNQCVYI YAKEMSYDEC FEKKSFVCQA DQWA

The invention further provides a gene encoding an accessory glandprotein identified as Acp32CD. The cDNA sequence for Acp32CD is asfollows (SEQ ID NO:8):

1 cagctgcgct gttgcttcag ctgactgcag caacatggac taacaactgc cactgttttt 61cagttcccca gatatgcccc ccttgttgcg gcactgcttt ggacacgcct ttatcggact 121gccccttttt aatgggcaag agcagccacg gccacagtca aatcgattcg attccgggca 181acggcggtca tctctatata taagggacgg tcggacagcc cgggcagctc agcgttgcag 241cgatgtggcg gatgcggatg cggctactca ctggctactt ggtcctgtgg ccctcggcca 301actgccggag catggcgctc tgggccagaa gtactacatg aactttgcct tcaataataa 361taatccggat ggcgaaggag gcaccggcgt cgatggtggc ggtggtggtg ctggtggtgg 421tgccgctggt cccggcggtg gaaccgggga ttcgccccat tcacaagaag gtgacggtag 481tgctgcgacg gataacccca atgacgacca cgctacatct gctgataata gtttggctac 541tgatggcgat gccattggta agaaggaaag cggcggtggt agcgatggca agagcgactc 601caaagactcg agcggaggca atgatgccac gccagcgaat ggtcatgacg atgacaacga 661tgacagcgac agaaggatgc caaggatcga caagataagg aagaggaggc cggacaggag 721gggaagcgca ccgatcacag ccatcacagt agctacgaga tcagatcgac gacagctttg 781gcgggcggta cgtgcggtcc atttacgaga gcagcgagag ccacggacat tcggcagcaa 841tgccggctcc aatcagcgga caatggagcc cgtgagagca gtcaggagaa ccaggatgcc 901aaggaagtgg ccagcgaagc gcctgctcaa cgggcaggca acgtgccaga tggacccgaa 961actggagcca accgggagat ctgaccacag gttcgctcga gggactttgg aagaacacag 1021acacttcaat tgattttatc attaaaatgc caataaaatt ttaataccaa aaaaaaaaaa 1081aaaaa

Two open reading frames are found within the nucleotide sequence of theAcp32CD cDNA. Open reading frames 1 and 2 are provided as SEQ ID NOS:9and 10, respectively, as follows:

1 atgcccccct tgttgcggca ctgctttgga cacgccttta tcggactgcc cctttttaat (SEQID NO:9) 61 gggcaagagc agccacggcc acagtcaaat cgattcgatt ccgggcaacggcggtcatct 121 ctatatataa gggacggtcg gacagcccgg gcagctcagc gttgcagcgatgtggcggat 181 gcggatgcgg ctactcactg gctacttggt cctgtggccc tcggccaactgccggagcat 241 ggcgctctgg gccagaagta ctacatgaac tttgccttca acaacaataatccggatggc 301 gaaggaggca ccggcgtcga tggtggcggt ggtggtgctg gtggtggtgccgctggtccc 361 ggcggtggaa ccggggattc gccccattca caagaaggtg acggtagtgctgcgacggat 421 aaccccaatg acgaccacgc tacatctgct gataatagtt tggctactgatggcgatgcc 481 attggtaaga aggaaagcgg cggtggtagc gatggcaaga gcgactccaaagactcgagc 541 ggaggcaatg atgccacgcc agcgaatggt catgacgatg acaacgatgacagcgacaga 601 aggatgccaa ggatcgacaa gataaggaag aggaggccgg acaggaggggaagcgcaccg 661 atcacagcca tcacagtagc tacgagatca gatcgacgac agctttggcgggcggtacgt 721 gcggtccatt tacgagagca gcgagagcca cggacattcg gcagcaatgccggctccaat 781 cagcggacaa tggagcccgt gagagcagtc aggagaacca ggatgccaaggaagtggcca 841 gcgaagcgcc tgctcaacgg gcaggcaacg tgccagatgg acccgaaactggagccaagg 901 aagatgacta cgaggagatg taacacaacc gggagatctg accacaggttcgctcgaggg 961 actttggaag aacgcagaca cttcaat

1 atgaactttg ccttcaataa taataatccg gatggcgaag gaggcaccgg cgtcgatggt (SEQID NO:10) 61 ggcggtggtg gtgctggtgg tggtgccgct ggtcccggcg gtagaaccggggattcgccc 121 cattcacaag aaggtgacgg tagtgctgcg acggataacc ccaatgacgaccacgctaca 181 tctgctgata atagtttggc tactgatggc gatgccattg gtaagaaggaaagcggcggt 241 ggtagcgatg gcaagagcga ctccaaagac tcgagcggag gcaatgatgccacgccagcg 301 aatggtcatg acgatgacaa cgatgacagc gacagaagga tgccaaggatcgacaagata 361 aggaagagga ggccggacag gaggggaagc gcaccgatta cagccatcacagtagctacg 421 agatcagatc gacgacagct ttgtgccggc tccaatcagc ggacaatggagcccgtgaga 481 gcagtcagga gaaccaggat gccaaggaag tggccagcga agcgcctgctcaacgggcag 541 gcaacgtgcc agatggaccc gaaactggag ccaaggaaga tgactacgaggagatgtaac 601 acaaccggga gatctgacca caggttcgct cgagggactt tggaagaacgcagacacttc 661 aat

Open reading frame 1, encodes a protein of 269 amino acids. Thepredicted amino acid sequence from open reading frame 1 of the ACP32CDgene is provided in SEQ ID NO:11, as follows:

1 MPPLLRHEFG HAFIGLPLFN GQEQPRPQMN FAFNNNNPDG EGGTGVDGGG GGAGGGAAGP 61GGGTGDSPHS QEGDGSAATD NPNDDHATSA DNSLATDGDA IGKKESGGGS DGKSDSKDSS 121GGNDATPANG HDDDNDDSDR RMPRIDKIRK RRPDRRGSAP ITAITVATRS DRROLWRAVR 181AVHLREOREP RTFGSNAGSN QRTMEPVRAV RRTRMPRKWP AKRLLNGQAT CQMDPKLEPR 241KMTTRRCNTT GRSDHRFARG TLEERRHFN

Open reading frame 2, encodes a shorter protein lacking the aminoterminal 28 amino acids of the protein encoded by open reading frame 1.The predicted amino acid sequence from open reading frame 2 of theACP32CD gene is provided in SEQ ID NO:12, as follows:

1 MNFAFNNNNP DGEGGTGVDG GGGGAGGGAA GPGGGTGDSP HSQEGDGSAA TDNPNDDHAT 61SADNSLATDG DAIGKKESGG GSDGKSDSKD SSGGNDATPA NGHDDDNDDS DRRMPRIDKI 121RKRRPDRRGS APITAITVAT RSDRROLWRA VRAVHLREOR EPRTEGSNAG SNQRTMEPVR 181AVRRTRMPRK WPAKRLLNGQ ATCQMDPKLE PRKMTTRRCN TTGRSDHRFA RGTLEERRHF

The present invention also provides a gene encoding an accessory glandprotein which is identified as Acp33A (SEQ ID NO:13):

1 ggattctaca agatgctacc ttccaagcga gttccatttc ttttcaccat tatcctgttt 61ctggctggac tgggtcagca cacaactgaa agtgtacttc cagactgcgt tctttatcca 121agatgtttga tcacaaagga tccgtgttgc atgtaaagca tctatagaaa tcaaagaagg 181atcttgtata tggaagtgga aatgtggttc aaaatatcag cacttctcaa ggctgtaaca 241aaaactgcac tctgttttta taaatacaaa tatccacaaa tatctggtct taacatggca 301acatttcaag tcttccaata aatcattttc gtttttattg tgaaaaaaaa aaaaaaaaaa 361aaaaaaa

Two open reading frames are also found within the nucleotide sequence ofthe Acp32CD cDNA. Open reading frames 1 and 2 are provided as SEQ IDNOS:14 and 15, respectively, as follows:

1 atgctacctt ccaagcgagt tccatttctt ttcaccatta tcctgtttct ggctggactg (SEQID NO:14) 61 ggtcagcaca caactgaaag tgtacttcca gactgcgttc gactgcgttctttatccaag 121 atgtttga tcacaaagga tccgtgttgc atg

1 atggaagtgg aaatgtggtt caaaatatca gcacttctca aggctgtaac aaaaactgca (SEQID NO:15) 61 ctctgttttt ataaatacaa atatccacaa atatctggtc ttaacatggcaacatttcaa 121 gtcttccaa

Open reading frame 1, encodes a protein of 47 amino acids. The predictedamino acid sequence from open reading frame 1 of the ACP32CD gene isprovided in SEQ ID NO:16, as follows:

1 MLPSKRVPFL FTIILFLAGL GQHTTESVLP DCVLYPRCLI TKDPCCM

Open reading frame 2, encodes a protein of 43 amino acids. The predictedamino acid sequence from open reading frame 2 of the ACP32CD gene isprovided in SEQ ID NO:17, as follows:

1 MEVEMWFKIS ALLKAVTKTA LCFYKYKYPQ ISGLNMATFQ VFQ

The present invention also provides a gene encoding an accessory glandprotein which is Acp36DE (SEQ ID NO:18):

1 caaatcaaca actaattaat gcacaagacg tgctctcaga caaagatcag aaacagacgc 61aagttcagaa caataactta catattcgat tcggtgtgtc agcactaaga gaaggaagaa 121ataatcctag tttggaaacg atttctcggg ataaagtaga taaaatatca cctgcattgc 181agttgcaact gttgagatat gcagattctc agtcgcaatc ccagacgcag tcacaatctg 241cctcacaatc tgaatcaaat gcatcttcac aattccaggc acaggagcaa agcaatcgac 301tgttgggaaa acccacctgt ttcagaatct cagtcacaat cagagtcaca gtcacagtcc 361gagtcacaga agcagtcaca gtcgcagtca cagcgacagc aacagataca gacgcaattg 421caaatactgc gacagttgca acaaaagtca aatgagcaat ctgccgcaca atctgcttct 481cagattcaat cgcagaggca atcggattct caatccaact tacaattaca agaacaatca 541caatcgcagt cagagcaagg taagccaatc cagtcacaaa ttcaaattct tcaagggctg 601cagcaaaaag agttagatga caaatctgca tcacagtcgc agtccgaatc caagacacgg 661aaagagcaac aaaaacagtt gaatttgcaa caacttgagg agctatcgtc ttcactatca 721cagtcacggc tagggctggg acagcaaatc cagtcacagc tacaaaagaa tcagttggat 781aagcaatttt cttcacagtt tcagtcacaa tccaagtcac agctggagca acaaatgcaa 841ttgcaattac aaagccttcg gcaactgcag cagaagcaat tagatgagca atctgcttca 901cagtcgcagc cacagtcaca ggtagcgcaa cagatccagt cacatttgca acttcttcga 961ttactgcaat ccagattgaa gacgcagtcg gcattgaaat cagatttaga acaacaaatc 1021ctttttcaat taaagaaact tacagaagtg caacagaaac agttggctga gcaacccacc 1081ttacgaccca gttcaaaatc acaatcgcct gggcagctag agcagcaaat tctgttacac 1141ctgcaaaatc ttctacactt tcagcagaat cagctaaaat cagatacaca aacccaaagc 1201cagttgcaag agtcaaaatc taactcactg tcacagtcac agtcacaatc gcaggagcag 1261ttacagttgc agcgggatca gaatcttcgg caattggaac aagtaaagtt ggaaatgcaa 1321aatattcgag agctgctgca gaagggcaag tctgagctac aaacccaatc ggactctcag 1381cgacgtatac atgagctata ccaaaatatt ctgcagctaa ataaggagaa gttgagctac 1441caattgaaac agttaaaact aaaagaattg gaagaccaaa agaagtcgca ggcagaaata 1501tcaaagggaa gtaacccatc caatctattt attatcggac aattgccttc cgaaggaaag 1561ccagctcctg gaaatcaagg tccttcaatt gagcctaagc tggtccccca acccggttca 1621ctggacaaat tgccatcagg cggagggcta attggcaagc cagcttcaac aggactgtat 1681attttatcgc cggatttcaa tgatttgtcg gattaccgag atcagtttcg tctacagcaa 1741gaattaaaaa agcatcaaaa tatattgagc cttttgcagc gtagacaaaa tgataaaaaa 1801caacaaaacg cacagctgtt gctaggacaa caacagaagg aacaacaagc tcaggaatca 1861atcaataaac aacagtcctc atctgctggc tctagttctc agaccaagtt acagcaagat 1921atacaaagta ctggagctca aggctcacag cagggtcttc aagctggatc cactggcctg 1981cagactagtt ccctacaagg cacagaaagt tctgcatctc aaagcgctct tcagcgattg 2041aaggagcagg aacaactgcg aattcagacg gaaaatgatc agaaaacctc ttcttcaagc 2101tcgcacagta actcacaaaa ctcgcagagt tcgtcatcac agtcatcgca ggcatcacag 2161tctgaagcac aacgacagga ggctggcaat cgaaatacct tgctactaga tcaatcgagc 2221tccaagactc agtcgagtcg aagtccgagt cgtcgtctca atcatcgtca cattcatcgt 2281cgcagtcaac gtcgaactca tcttcaaacg ttcaatcgaa actacaagga gaaagccaag 2341cgctgctaaa caatttgtca ggttaagtag ttaaccttat acttctcaca gtactgacat 2401gggcgaagag cagccttatt cagatgttaa tcaaaaagag gaaataaaat aattgttctt 2461tcatttaaaa actcgaaaaa aaaaaaaaaa aa

An open reading frame encoding the Acp36DE protein is found within thecDNA sequence (SEQ ID NO:19):

1 atgcagattc tcagtcgcaa tcccagacgc agtcacaatc tgcctcacaa tctgaatcaa 61atgcatcttc acaattccag gcacaggagc aaagcaatcg actgttggga aaacccacct 121gtttcagaat ctcagtcaca atcagagtca cagtcacagt ccgagtcaca gaagcagtca 181cagtcgcagt cacagcgaca gcaacagata cagacgcaat tgcaaatact gcgacagttg 241caacaaaagt caaatgagca atctgccgca caatctgctt ctcagattca atcgcagagg 301caatcggatt ctcaatccaa cttacaatta caagaacaat cacaatcgca gtcagagcaa 361ggtaagccaa tccagtcaca aattcaaatt cttcaagggc tgcagcaaaa agagttagat 421gacaaatctg catcacagtc gcagtccgaa tccaagacac ggaaagagca acaaaaacag 481ttgaatttgc aacaacttga ggagctatcg tcttcactat cacagtcacg gctagggctg 541ggacagcaaa tccagtcaca gctacaaaag aatcagttgg ataagcaatt ttcttcacag 601tttcagtcac aatccaagtc acagctggag caacaaatgc aattgcaatt acaaagcctt 661cggcaactgc agcagaagca attagatgag caatctgctt cacagtcgca gccacagtca 721caggtagcgc aacagatcca gtcacatttg caacttcttc gattactgca atccagattg 781aagacgcagt cggcattgaa atcagattta gaacaacaaa tcctttttca attaaagaaa 841cttacagaag tgcaacagaa acagttggct gagcaaccca ccttacgacc cagttcaaaa 901tcacaatcgc ctgggcagct agagcagcaa attctgttac acctgcaaaa tcttctacac 961tttcagcaga atcagctaaa atcagataca caaacccaaa gccagttgca agagtcaaaa 1021tctaactcac tgtcacagtc acagtcacaa tcgcaggagc agttacagtt gcagcgggat 1081cagaatcttc ggcaattgga acaagtaaag ttggaaatgc aaaatattcg agagctgctg 1141cagaagggca agtctgagct acaaacccaa tcggactctc agcgacgtat acatgagcta 1201taccaaaata ttctgcagct aaataaggag aagttgagct accaattgaa acagttaaaa 1261ctaaaagaat tggaagacca aaagaagtcg caggcagaaa tatcaaaggg aagtaaccca 1321tccaatctat ttattatcgg acaattgcct tccgaaggaa agccagctcc tggaaatcaa 1381ggtccttcaa ttgagcctaa gctggtcccc caacccggtt cactggacaa attgccatca 1441ggcggagggc taattggcaa gccagcttca acaggactgt atattttatc gccggatttc 1501aatgatttgt cggattaccg agatcagttt cgtctacagc aagaattaaa aaagcatcaa 1561aatatattga gccttttgca gcgtagacaa aatgataaaa aacaacaaaa cgcacagctg 1621ttgctaggac aacaacagaa ggaacaacaa gctcaggaat caatcaataa acaacagtcc 1681tcatctgctg gctctagttc tcagaccaag ttacagcaag atatacaaag tactggagct 1741caaggctcac agcagggtct tcaagctgga tccactggcc tgcagactag ttccctacaa 1801ggcacagaaa gttctgcatc tcaaagcgct cttcagcgat tgaaggagca ggaacaactg 1861cgaattcaga cggaaaatga tcagaaaacc tcttcttcaa gctcgcacag taactcacaa 1921aactcgcaga gttcgtcatc acagtcatcg caggcatcac agtctgaagc acaacgacag 1981gaggctggca atcgaaatac cttgctacta gatcaatcga gctccaagac tcagtcgagt 2041cgaagtccga gtcgtcgtct caatcatcgt cacattcatc gtcgcagtca acgtcgaact 2101catcttcaaa cgttcaatcg aaactacaag gagaaagcca agcgctgc

The predicted amino acid sequence of the Acp36DE protein is as follows(SEQ ID NO:20):

1 MQILSRNPRR SHNLPHNLNQ MHLHNSRHRS KAIDCWENPP VSESQSQSES QSQSESQKQS 61QSQSQRQQQI QTQLQILRQL QQKSNEQSAA QSASQIQSQR QSDSQSNLQL QEQSQSQSEQ 121GKPIQSQIQI LQGLQQKELD DKSASQSQSE SKTRKEQQKQ LNLQQLEELS SSLSQSRLGL 181GQQIQSQLQK NQLDKQFSSQ FQSQSKSQLE QQMQLQLQSL RQLQQKQLDE QSASQSQPQS 241QVAQQIQSHL QLLRLLQSRL KTQSALKSDL EQQILFQLKK LTEVQQKQLA EQPTLRPSSK 301SQSPGQLEQQ ILLHLQNLLH FQQNQLKSDT QTQSQLQESK SNSLSQSQSQ SQEQLQLQRD 361QNLRQLEQVK LEMQNIRELL QKGKSELQTQ SDSQRRIHEL YQNILQLNKE KLSYQLKQLK 421LKELEDQKKS QAEISKGSNP SNLFIIGQLP SEGKPAPGNQ GPSIEPKLVP QPGSLDKLPS 481GGGLIGKPAS TGLYILSPDF NDLSDYRDQF RLQQELKKHQ NILSLLQRRQ NDKKQQNAQL 541LLGQQQKEQQ AQESINKQQS SSAGSSSQTK LQQDIQSTGA QGSQQGLQAG STGLQTSSLQ 601GTESSASQSA LQRLKEQEQL RIQTENDQKT SSSSSHSNSQ NSQSSSSQSS QASQSEAQRQ 661EAGNRNTLLL DQSSSKTQSS RSPSRRLNHR HIHRRSQRRT HLQTFNRNYK EKAKRC

The present invention provides a gene encoding an accessory glandprotein which is Acp53Ea (SEQ ID NO:21):

1 ccgaaagcac acagataagg cttccaatga aactgataaa ggttacacta gtgttcagct 61tactggctct cgtatttgtg gcccaaacgg aggcgcaaaa tccaatatgg gagaattggc 121tggcatgcaa tagaattggt actaaagcgc ttgccagtct gctgagagaa acaattccaa 181ccgttcgtaa tttactgaac tgcattgact tcaatccacc aaccgatatt ggaaatagtt 241acctttcaaa acttaagtta tactatgagc ttgttaagcg aggtgcgctt gacaagactc 301agtgtctgat tgtgccactc aaggaatcag tgagactact gaggccttat gtaaaatcgc 361ttgagaccaa caaatgcttg ggtgaataaa tcactatttt ggccatagta aaataaattt 421ctgagcatta ataaagcacg

An open reading frame encoding the Acp53Ea protein is found within thecDNA sequence (SEQ ID NO:22):

1 atgaaactga taaaggttac actagtgttc agcttactgg ctctcgtatt tgtggcccaa 61acggaggcgc aaaatccaat atgggagaat tggctggcat gcaatagaat tggtactaaa 121gcgcttgcca gtctgctgag agaaacaatt ccaaccgttc gtaatttact gaactgcatt 181gacttcaatc caccaaccga tattggaaat agttaccttt caaaacttaa gttatactat 241gagcttgtta agcgaggtgc gcttgacaag actcagtgtc tgattgtgcc actcaaggaa 301tcagtgagac tactgaggcc ttatgtaaaa tcgcttgaga ccaacaaatg cttgggtgaa

The predicted amino acid sequence of the Acp53Ea protein is as follows(SEQ ID NO:23):

1 MKLIKVTLVF SLLALVFVAQ TEAQNPIWEN WLACNRIGTK ALASLLRETI PTVRNLLNCI 61DFNPPTDIGN SYLSKLKLYY ELVKRGALDK TQCLIVPLKE SVRLLRPYVK SLETNKCLGE

The present invention provides a gene encoding an accessory glandprotein which is identified as Acp63F (SEQ ID NO:24):

1 ctttgcaaga tgaaagctat catcgttttt attctgttca tttcaagtgt gcatgctatg 61agcaaatgca accaagcaat ttatctaaat cttgatcctc actgcggaat acttcccgat 121tgtaacttag atggtccaaa tccaagttac ctcaataggg tgtcgtgtga acgcaaagaa 181aacggaaaac caggattcat cgaactaatt cccggaaaat gtctccatgg taaaccgcgt 241tgctcgttaa aatagtaata ttgttccaat atttccatgc atatatgttt caattaaagg 301cattataaat acctataaaa aaa

An open reading frame encoding the Acp63F protein is found within thecDNA sequence (SEQ ID NO:25):

1 atgaaagcta tcatcgtttt tattctgttc atttcaagtg tgcatgctat gagcaaatgc 61aaccaagcaa tttatctaaa tcttgatcct cactgcggaa tacttcccga ttgtaactta 121gatggtccaa atccaagtta cctcaatagg gtgtcgtgtg aacgcaaaga aaacggaaaa 181ccaggattca tcgaactaat tcccggaaaa tgtctccatg gtaaaccgcg ttgctcgtta 241aaa

The predicted amino acid sequence of the Acp63F protein is as follows(SEQ ID NO:26):

1 MKAIIVFILF ISSVHAMSKC NQAIYLNLDP HCGILPDCNL DGPNPSYLNR VSCERKENGK 61PGFIELIPGK CLHGKPRCSL K

The present invention provides a gene encoding an accessory glandprotein which is identified as Acp76A (SEQ ID NO:27):

1 atatcaagct tatgactctt ggataagcca ttgatatagc aattgtaaat atattatgtg 61caattctcct tattttctaa gacattctta ataatataat gcgaactaag gttacattcc 121atctgcgcat gcgtgagtcc ttttgctcag caggaacgaa agtacaacgg gtcgtatgag 181ttatggcaat cgaatgggca cacgtggccg ccggtccaat ggccagtaaa tcaaactttt 241tggcagggcg agtaaacagt gttataaatc agaaaaccgc aaggcagcca ccaggcagct 301accatattgt ccatggagga aagcagacag ttggcagact taagtcggac gaaaagacat 361ccacccacgc gggcggattg caatgtgcca ggatgcaagt gacgcatagt gctattaaca 421tattaccagg cggtaagtgg gtggataaca cattcagtcg gtggatggaa gtacgtgcat 481agaacataga gctgccagtg aatattggag caattggagc actgggtgct aaatatgagc 541acttgatgaa aacataacac tgactaaaca agatatttct ctgcggctga atttatctag 601cgaaagtgtg aaattgttgt tgatttatgt tatactaagc acgccatata tatgaggtgg 661cgaatttacg acgatttact aagattttaa tgtactcttc cttggttaga ggatagaaag 721catgaaattg aatagcagtg caataaatcc gtactaattt ccattcgttt ttgcgtatta 781tccaatatct taagggtcat tccccttgtg tgtatatata ggaacaaagt aagtttaaga 841agctcattgc ggctttggaa aatgggcaac catcaagtaa cattcttagt actgtgcacg 901tcgctcctct ttcaaaatac aatacaacaa aatgtatcat ttcaactgat aagggaaatc 961gatagataca caccagagaa ttttgtacta tcagtgttga atatagaaat gattcttttt 1021gagatccatg ccgctaaggc agttgaaagt aataacgatt tggaaaggag cttgatcata 1081aactttggat actccgaagc aaggcaggaa gtactggatt ggggattgag atataagaaa 1141gcctcgagcg ccaagttcca gatggccaac aaggtggcag tgtctcagaa actgccccta 1201tcgcaaaagc tgcgtctggt aaacgaggtg ctgatgacga gcgccaagaa gtatgatgta 1261acaaaggatg tcagaccatc aaaattaatg gatgaatggt tgtcctccca tttggatggt 1321gtactcgcca attttgtaca agagaagaag ttaaacgcgg gcgaaaacat tgtagccatc 1381agcggaatga cagtcactcc cctttgggca tctcatttcc aatcagagat taatagatac 1441tttgtcaata atcctggcac tggatatgct tcgaaagacc caacatgtgt gcccatgatg 1501cactcattgt cctcgtttga aaccatgtcc acggacgagg ccaaaggtat atacattcca 1561ttctcatcgg caaacttggg tatgttgatc ctcctgccga ggaaaggtgt cacctgcaag 1621gacattttgg ataatttaaa caaccagatc aatgtggaat ataatgatca caaggatgtt 1681cacttgctac tgcccatatt caaggagaaa tttgactaca atattgccaa attctttaac 1741ggaattaaca ttgaagacac gtttaaagat tcggcgttta aatcgaaagc caaaatcaaa 1801atcaacaact tccgagtcaa ccatggcata cgatttcaac ccattctccg tttagaagta 1861gttgatgata ttaatactgg aaagaccgaa acgtttgaag taaatcgccc atttgtcttt 1921gtcataaagg ataaggttaa cgtatacgca gttggtcgaa ttgaaaacct agatggactt 1981actgacaaag tgaattgctc caagaaatac gctgatctca agtcgtaaaa tatccataat 2041atatttcgaa gcataataaa gcaagaaaat ataaaaaaaa aaaaaaaaaa aa

An open reading frame encoding the Acp76A protein is found within thecDNA sequence (SEQ ID NO:28):

1 atgggcaacc atcaagtaac attcttagta ctgtgcacgt cgctcctctt tcaaaataca 61atacaacaaa atgtatcatt tcaactgata agggaaatcg atagatacac accagagaat 121tttgtactat cagtgttgaa tatagaaatg attctttttg agatccatgc cgctaaggca 181gttgaaagta ataacgattt ggaaaggagc ttgatcataa actttggata ctccgaagca 241aggcaggaag tactggattg gggattgaga tataagaaag cctcgagcgc caagttccag 301atggccaaca aggtggcagt gtctcagaaa ctgcccctat cgcaaaagct gcgtctggta 361aacgaggtgc tgatgacgag cgccaagaag tatgatgtaa caaaggatgt cagaccatca 421aaattaatgg atgaatggtt gtcctcccat ttggatggtg tactcgccaa ttttgtacaa 481gagaagaagt taaacgcggg cgaaaacatt gtagccatca gcggaatgac agtcactccc 541ctttgggcat ctcatttcca atcagagatt aatagatact ttgtcaataa tcctggcact 601ggatatgctt cgaaagaccc aacatgtgtg cccatgatgc actcattgtc ctcgtttgaa 661accatgtcca cggacgaggc caaaggtata tacattccat tctcatcggc aaacttgggt 721atgttgatcc tcctgccgag gaaaggtgtc acctgcaagg acattttgga taatttaaac 781aaccagatca atgtggaata taatgatcac aaggatgttc acttgctact gcccatattc 841aaggagaaat ttgactacaa tattgccaaa ttctttaacg gaattaacat tgaagacacg 901tttaaagatt cggcgtttaa atcgaaagcc aaaatcaaaa tcaacaactt ccgagtcaac 961catggcatac gatttcaacc cattctccgt ttagaagtag ttgatgatat taatactgga 1021aagaccgaaa cgtttgaagt aaatcgccca tttgtctttg tcataaagga taaggttaac 1081gtatacgcag ttggtcgaat tgaaaaccta gatggactta ctgacaaagt gaattgctcc 1141aagaaatacg ctgatctcaa gtcg

The predicted amino acid sequence of the Acp76A protein is as follows(SEQ ID NO:29):

1 MGNHQVTFLV LCTSLLFQNT IQQNVSFQLI REIDRYTPEN FVLSVLNIEM ILFEIHAAKA 61VESNNDLERS LIINFGYSEA RQEVLDWGLR YKKASSAKFQ MANKVAVSQK LPLSQKLRLV 121NEVLMTSAKK YDVTKDVRPS KLMDEWLSSH LDGVLANFVQ EKKLNAGENI VAISGMTVTP 181LWASHFQSEI NRYFVNNPGT GYASKDPTCV PMMHSLSSEE TMSTDEAKGI YIPFSSANLG 241MLILLPRKGV TCKDILDNLN NQINVEYNDH KDVHLLLPIF KEKFDYNIAK FFNGINIEDT 301FKDSAFKSKA KIKINNFRVN HGIRFQPILR LEVVDDINTG KTETFEVNRP FVFVIKDKVN 361VYAVGRIENL DGLTDKVNCS KKYADLKS

The present invention provides a gene encoding an accessory glandprotein which is identified as Acp98AB (SEQ ID NO:30):

1 atggaattcc ctaatcctgt tcttagaaga gcagcaggac attccgaacg gaaaggctac 61acacactatc aaaggatgac gaggatgtcc aagtgaatca ccagctaaag caaggatatt 121atgtatatct aaggaaatca aataaacttg catgctctaa aaa

An open reading frame encoding the Acp98AB protein is found within thecDNA sequence (SEQ ID NO:31):

1 atggaattcc ctaatcctgt tcttagccgc attgggcgca gcctccgcac gaataagggg 61acacactatc aaaggatgac gaggatgtcc aag

The predicted amino acid sequence of the Acp98AB protein is as follows(SEQ ID NO:32):

1 MEFPNPVLSR IGRSLRTNKG THYQRMTRMS K

The present invention also provides a method for determining whether afemale Drosophila melanogaster has recently mated. An antibody, fragmentthereof, or probe which recognizes an acessory protein, including thoseproteins described in SEQ ID NO:4, SEQ ID NO:7, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:20, SEQ ID NO:23, SEQ IDNO:26, SEQ ID NO:29, and SEQ ID NO:32, is used to identify the presenceof the accessory gland protein in the female after mating. The antibody,fragment thereof, or probe is bound to a label so that it is effectiveto permit detection of the protein upon binding of the antibody to theprotein. The labeled antibody is contacted with a fluid or tissue samplefrom the female Drosophila melanogaster under conditions effective topermit binding of the antibody to the protein. The presence of any ofprotein in the biological sample by is determined by detecting thelabel.

EXAMPLES Example 1 Materials and Methods

Fly Handling and Rearing

Wild type Canton S or Oregon R D. melanogaster were maintained onyeast-glucose media at room temperature. Unmated animals wereanesthetized with CO₂ and collected within 10 hours of eclosion. Flieswere kept in fresh vials isolated from the opposite sex until dissectedor quick-frozen prior to RNA extraction.

Isolation of New Accessory Gland Genes.

By minor modifications of procedures described in DiBenedetto (1987),32P-labeled cDNAs were prepared from the poly A+ RNA of 2-day-old adultfemale Canton S flies and, separately, from accessory glands (attachedto ejaculatory ducts) that had been dissected from 2-day-old adult maleCanton S flies. These cDNAs were hybridized to duplicate sets of filterscontaining a total of 22,000 clones of genomic DNA in Charon 4A(Maniatis, 1978). One set of filters received 1.2×10⁷ cpm of labeledcDNA from male reproductive tissues. Prior to incubation with thefilters this cDNA was prehybridized with 20 ug of polyA+ RNA from adultfemales; the female RNA competitor was left in during the filterhybridization. The second set of filters received 1.2×107 cpm offemale-derived cDNA, without competitor.

39 clones that consistently showed strong (or exclusive) hybridizationto the male tissue probe but not to the female tissue probe throughthree sequential rounds of plaque purification and screening wereretained for further study. These clones were then screened forhybridization to RNAs from accessory glands, or ejaculatory ducts, ortestes, or the remainder of the male, or whole adult females. A clone isdefined as encoding an accessory gland-specific transcript if that RNAis present in accessory glands but not in any of the other RNA sourceslisted above. Twenty clones fell into this category. By screening these20 clones for hybridization to previously cloned accessory gland genes,six re-isolates of the genomic region encoding Acp95EF were found(DiBenedetto, 1987; DiBenedetto, 1990). The remaining 14 clonescorresponded to previously-unreported Acp genes. Of these clones, threeencompassed overlapping segments of genomic DNA encoding Acp33A; theremaining 11 were single isolates.

Nucleic Acids

The fragment(s) of each genomic clone homologous to the male RNA wereidentified by Northern and “reverse Northern” strategies. Thosefragments were used as probes to isolate cDNA clones from male adultDrosophila melanogaster libraries (Poole, 1985; Monsma and Wolfner,1988). Routine nucleic acid manipulations including cloning andsubcloning into Bluescript vectors (Stratagene), restriction mapping,Southern blotting, determination of transcriptional orientation, DNAsequencing by the dideoxy chain termination method (Sanger, 1977), andisolation of overlapping genomic clones for Acp36DE were doneessentially as in Maniatis (1978) and Ausubel (1994). RNA preparation,Northern blotting and hybridizations in situ to polytene chromosomeswere by slight modifications of methods described in DiBenedetto (1987).When needed, sequences from multiple clones of a given Acp were alignedto obtain the complete sequence of the open reading frame. In fourinstances (Acps 32CD, 33A, 76A and 98AB) the cDNA clone was incompleteand lacked the start of the open reading frame and/or any untranslatedleader region. Since our focus is on the protein encoded by the Acpgene, RACE-PCR and/or sequencing of genomic DNA immediately 5′ to thefirst base of the cDNA clone was used to obtain the full open readingframe. This analysis did not extend to defining the starting nucleotideof the Acp mRNA; thus lengths of 5′ UTRs given in FIG. 1 are minimalsizes. Sequences were analyzed with GCG sequence analysis software, andhave been submitted to GenBank. Accession numbers are given in FIG. 1.

Whole-mount in Situ Hybridization

Accessory glands were dissected in PBS and treated essentially as inLehmann and Tautz (1994), except that PBS was used rather than PBST,pretreated the tissue in 50 ug/ml of proteinase K followed by incubationin 2 mg/ml glycine in PBS-Triton, and eliminated heparin from thehybridization buffer. Hybridization and detection with adigoxigenin-labeled probe (Genius Kit, Boehringer Mannheim) was by minormodification of the procedures in Tautz and Pfeifle (1989). Stainedaccessory glands were mounted on slides in 25% glycerol and photographedusing a Zeiss Axioskop and Kodak Gold 200 film.

Example 2 New Genes Encoding Accessory Gland Proteins (Acps)

In differential cDNA hybridization screens, 12 independent new geneswere identified (FIG. 1). The genomic clones were restriction-mapped,and their fragment(s) that hybridized to the accessory gland-specifictranscript were determined. On Southern blots of genomic DNA, 11 of thegenes showed hybridization patterns consistent with single copy genes.All but one of these genes hybridized in situ to a single polytene band;the exception was Acp33A, which showed a minor second site ofhybridization at 32E. Each gene's chromosomal position is included inthe gene's name in accordance with Drosophila melanogaster nomenclaturerules. Two genes, Acp53Ea and Acp53Eb, are derived from polytene band53E. However they are distinct, non cross-hybridizing genes whoseimmediate chromosomal regions do not overlap. The genomic clonescontaining Acp genes did not hybridize to any other male-limitedtranscripts. However, some of the genomic clones contained regions thathybridized additionally to sex-nonspecific or female-specifictranscripts.

The twelfth gene detected in the screens, Acp rep1, had characteristicsof a moderately-repetitive gene. Acp rep1 probes hybridized to multiplebands on genomic Southern blots and multiple polytene loci upon in situhybridization. This gene was not studied further.

On the basis of their chromosomal positions and/or hybridization, thesenew genes do not correspond to any of the previously-reported D.melanogaster Acp genes identified in hybridization screens or frompurified accessory gland peptides (Schäfer, 1986; DiBenedetto, 1987;Chen, 1988; Monsma and Wolfner, 1988; Simmerl, 1995). One of the 11unique genes, Acp36DE, may correspond to a previously-identified locus,AcpC, which was detected via a natural variant lacking a protein band of˜125-128 kDa on SDS polyacrylamide gels of accessory gland proteins(Whalen and Wilson, 1986). As described below, Acp36DE encodes a proteinof a predicted molecular weight of 81.3 kDa. However, the protein'ssequence contains a large number of potential and actual (Bertram, 1996)glycosylation sites, and antibodies against Acp36DE recognize a 122 kDaglycoprotein (Bertram, 1996). Unfortunately, the AcpC variant strainsreported by Whalen and Wilson (1986) were no longer extant when Acp36DEwas discovered, so it is impossible to determine whether AcpCcorresponds to Acp36DE. The approximate genetic map position of oneother Acp gene reported by Whalen and Wilson (1986), AcpB, is near Acps29AB, 31F and 32CD. The other two positions reported by Whalen andWilson (1986) do not correspond to sites of any of the Acp genesidentified.

Thus, 19 non-repetitive Acp genes have been cloned (Schäfer, 1986;DiBenedetto, 1987; Chen, 1988; Monsma and Wolfner, 1988; Simmerl, 1995;and this study). These genes represent 16 independent chromosomalregions; 26A and 57D contain clusters of two (Acp26Aa,b; Monsma andWolfner, 1988) and three (57Da,b,c; Simmerl, 1995) unrelated Acp genes,respectively.

Interestingly, the new nonrepetitive Acp genes are all autosomal. So areall four Acp genes or gene clusters isolated in previous screens thatwere unbiased as to chromosomal position (Schäfer, 1986; DiBenedetto,1987; Chen, 1988). Given that the X chromosome represents about 20% ofthe genome, the probability that all 15 Acp gene regions would falloutside the X is (0.8) 15, which equals 0.035. Even making worst-caseassumptions about the library screened (Maniatis, 1978), like itscontaining no overlapping clones and its deriving from DNA of a 50-50mixture of XX and XY embryos, it is calculated that p=0.076, which ishighly suggestive of nonrandom placement of Acp genes. The absence ofAcp genes from the X chromosome might relate to their male-limitedexpression. In D. melanogaster males, autosomal genes are present in twocopies per cell whereas X-linked genes occur in only one copy per cell.Dosage compensation increases the transcription of non sex-specificX-linked genes in male cells so that their transcript levels areequivalent to those produced by two copies of the gene female cells(reviewed in Kelley and Kuroda, 1995). It may be that autosomalplacement of Acp genes was advantageous since the genes could beexpressed at high levels without also needing to acquire dosagecompensation regulation.

Acp genes were first isolated in screens for male-specific transcripts(Schäfer, 1986; DiBenedetto, 1987). Not surprisingly, the screenreported here, with its enriched probe used in the presence of RNAcompetitor, was 10-fold more efficient than the earlier screens indetecting Acp genes. Considering that from this screen 10 genes wereisolated singly, three independent isolates of one were obtained, andsix were presumed independent isolates of another, a rough estimate ofthe number of Acp genes that can be isolated by this type of screen canbe made. Since one cannot assume equal representation of all sequencesin the library, Bunge and Fitzpatrick (1993) recommend using the methodof Esty (1986) for this estimate, even though this estimator isassociated with large standard deviations. This estimator[c^=c/(1−(c1/n)); Esty, 1986] predicts the existence of 25.3 Acp genesdetectable by our screening procedures. The method of Good (1950)predicts about 19 Acp genes detectable by the present procedures. Thismethod assumes all genes are equally represented in the library, basedon estimates of “coverage” as defined by Bunge and Fitzpatrick (1993).The Good method tends to yield an underestimate if the assumption ofcomplete coverage is not met. Though these are rough estimates, bothcalculations suggest that the Acp genes isolated in differential screensperformed here and by others represent more than half the genes thatcould be isolated in such screens, which will fail to detect rare RNAsor Acp genes flanked by highly-expressed non sex-specific transcriptionunits.

Example 3 Expression of the New Acps

Northern blot analysis was performed with each of the Acp genes; anexample of the results is shown in FIG. 2A. All the genes with theexception of Acp31F encode single accessory gland-specific transcripts.The transcripts fell into three size classes. Two genes encodedtranscripts larger than 2 kb, four encoded mRNAs between 0.95 and 2 kb,and six encoded RNAs smaller than 0.75 kb. The sizes of the transcriptsare listed in FIG. 1.

To determine the type of accessory gland cells in which thesetranscripts are produced, digoxigenin-labeled DNA probes complementaryto the transcripts of Acps 29AB, 31F, 33A, 36DE, 53Ea, 63F, 76A or 98ABwere hybridized in situ to RNA in whole mount accessory glands. In allcases, the probes hybridized strongly to mRNA in main cells (e.g. FIG.2B for Acp36DE). In most cases the strong staining of main cells made itimpossible to determine unambiguously whether there was also staining inthe secondary cells. In the case of Acp36DE an accessory glandfortuitously positioned so that secondary cells were visible at the edgeof the lobe was observed. In that case, it was possible to determinethat the secondary cells were unstained (FIG. 2B, inset). Geneexpression in main cells is stimulated in response to mating (Schmidt,1985; DiBenedetto, 1990; Monsma, 1990; Bertram, 1992; Simmerl, 1995).This stimulation is also observed for the one new gene tested, Acp36DE(Bertram, 1994).

Example 4 General Features of the Predicted New Acps

The goal in isolating new Acp genes was to identify new molecules thatcould ultimately be correlated with the functions of the accessorygland. Therefore, the sequence of the predicted proteins encoded by 9 ofthe new genes was determined.

Of the two Acp genes with large transcripts, the one with the simplertranscription pattern, Acp36DE, was selected for sequence analysis.cDNAs were sequenced from all the smaller Acp transcripts for which cDNAclones were cloned (Acps 29AB, 33A, 32CD, 53Ea, 62F, 63F, 76A, 98AB).FIG. 3 reports the open reading frame sequence of each Acp, with FIG. 1giving some of the salient numerical characteristics of the transcriptand its open reading frame. In all cases but two, only a single longopen reading frame in the Acp gene's transcript, beginning at the most5′ AUG of the mRNA, was found. The two exceptions were Acps 33A and36DE. The former encodes two open reading frames of similar size andcodon bias; it is not known which (or if both) is the bona fide Acp ORF.For Acp36DE, the first AUG initiates a short open reading frame of 18amino acids upstream of a 716 amino acid open reading frame. Antibodiesraised against the protein predicted by the long open reading frameconfirm that it is the expressed Acp (Bertram, 1996). As shown in FIG.1, 4 bases prior to the initiating AUGs of all the Acp genes listedshowed only partial matches to consensus for translational initiation inDrosophila (C/A A A C/A; Cavener, 1987). No consensus in these bases wasseen among Acp genes.

Several features are apparent in the new predicted Acps (FIG. 3). First,all except Acp36DE contain predicted secretion signals. A sequence thatmatches a consensus for signal sequence cleavage (von Heijne, 1983) isfound at the positions indicated in FIG. 3. Consistent with thisobservation, all Acps tested (29AB, 32CD, 53Ea, 62F, 63F and 98AB;Y.O.L., U.T. and M.F.W. unpublished, and 76A; Coleman, 1995) aresecreted and transferred to females during mating. Despite its lack ofan apparent signal sequence, Acp36DE is also secreted and transferred tofemales (Bertram, 1996). The predicted stretch of hydrophobic residuesat its N-terminus may function as a secretion signal.

The second feature of the predicted Acps is that many contain sequencesthat suggest they may be subject to post-translational modificationssuch as cleavage, amidation and glycosylation. Basic amino acids incontexts favorable for cleavage to liberate peptide hormones from aprecursor molecule are found in Acps 29AB, 32CD, 36DE, 53Ea, and 76A.Park and Wolfner (1995) have shown that cleavage of Acp26Aa occurs atsites consistent with the use of basic amino acids in such contexts(Schwartz, 1986; Benoit, 1987; Nakayama, 1992). Acp32CD contains apotential amidation site (173GKK; Kreil, 1984; Bradbury and Smyth,1987). Acps 29AB, 32CD, 36DE, 53Ea, 62F, 63F and 76A contain sites forpotential glycosylation (Kornfeld and Kornfeld, 1985; Miletich andBroze, 1990). The case of Acp36DE is particularly remarkable: inaddition to a site for N-linked glycosylation (Asn439-Pro-Ser) and infact being N-glycosylated (Bertram, 1996), Acp36DE contains largeamounts of serine and threonine, at which O-linked glycosylation couldoccur. These residues are mostly concentrated in four regions: aminoacids 42-64, 92-118, 334-351 and 591-665 of this glutamine-rich protein.Acp36DE also contains, along with Acps 32CD and 62F, sequences matchingconsensus for glycosaminoglycan attachment sites (Hassell, 1986;Bourdon, 1987), suggesting that these proteins could become associatedwith, or part of, an extracellular matrix. Taken together, thesefeatures suggest that the new Acps are secreted molecules, and some havethe potential to be cleaved to smaller peptides and/or be glycosylated.The Acps that do not contain cleavage sites (Acps 33A, 62F 63F, 98AB)are within the size range of known secreted accessory gland peptides(e.g. Chen, 1988; Monsma and Wolfner, 1988) and thus might besynthesized and secreted without further modification.

Example 5 Intriguing Sequence Similarities in Two Predicted Acps

The databases were searched for sequences that might have similaritiesto the 9 new predicted Acps. Two showed sequence similarity to proteinsin the data bases (FIGS. 3, 4).

Acp76A: A search of protein databases reveals that the 11 amino acidsstarting at position 345 and ending at 355, FEVNRPFVFVI (SEQ ID NO:33),are a perfect match to the serpin signature sequence,[LIVMF]-X-[LIVMFA]-[DNQ]-[RKHQ]-[PS]-F-[LIVMFY]-2X-[LIVMF] (SEQ IDNO:34) (Henikoff and Henikoff, 1994). This amino acid sequencesimilarity places the Acp76A in the serpin (serine protease inhibitors)superfamily of proteins. Members of this superfamily includealpha-1-antitrypsin, angiotensinogen, ovalbumin, antiplasmin, placentalplasminogen activator inhibitor, thyroxin binding protein, and heparincofactor II (Carrell and Boswell, 1986; Kanost, 1989). Many members ofthe serpin superfamily are extracellular serine protease inhibitors,helping to control proteolytic events associated with a wide variety ofregulated biochemical pathways (Carrell and Boswell, 1986; Gettins,1992; Zou, 1994). Several mammalian proteins in the serpin superfamilyplay roles in blood coagulation. Others are involved in fibrinolysis,inflammation, and tumor suppression. In humans a serpin, Protein CInhibitor (PCI), is present in semen and interacts with at least threedifferent proteases: urokinase plasminogen activator, tissue-typeplasminogen activator, and prostate specific antigen (Espana, 1993). Thephysiological role of these interactions in human semen is unknown.

Although it is not yet known whether Acp76A, with its perfect serpinsignature, is an active protease inhibitor, there are indications thatprotease inhibitors could play a critical role in controlling Acpfunction in D. melanogaster. Regulated proteolysis is a hallmark ofAcp26Aa's fate in mated female flies (Monsma, 1990; Park and Wolfner,1995). Acp36DE is also converted to a smaller form upon transfer tofemales (Bertram, 1996). Acp26Aa remains intact in the male'sreproductive tract. It undergoes ordered proteolysis in the genitaltract of the mated female, due to a combination of seminal fluidcomponents donated by the male as well as components donated by thefemale (Park and Wolfner, 1995). It is possible that the protease(s)that cleaves Acp26Aa is made by the male's accessory gland, but is keptin check in the gland by protease inhibitors such as, potentially,Acp76A or the protease inhibitor found in D. funebris accessory glands(Schmidt, 1989). Upon transfer to the female, the protease inhibitor(s)would be inactivated, allowing the protease(s) to become active and tocleave Acp26Aa.

A second possible role for a protease inhibitor derived from accessoryglands is in the coagulation of semen after mating. In Drosophila, as inmany other insects, a mating plug consisting of coagulated semen formsin the mated female's uterus. The plug is thought to facilitate thestorage of sperm by the female (Lefevre and Jonsson, 1962; Fowler, 1973;Bertram, 1996). By analogy to the role of members of the serpinsuperfamily in the tightly regulated proteolytic cascades that causeblood clotting in mammals (Gettins, 1992; Espana, 1993; Potempa, 1994),a serpin, potentially Acp76A, could function to regulate proteolyticcleavage events crucial to the formation of the mating plug. Consistentwith this suggestion, Acp76A is transferred to the female fly duringmating, and the majority of the transferred Acp76A is found in themating plug when it is expelled from the female between 2-4 hours aftermating (Coleman, 1995).

Acp62F: Acp62F has a 28 amino acid region of sequence similarity withseveral small neurotoxins made by the Brazilian “armed” spider,Phoneutria nigriventer. Sequence similarity is highest to the 48 aminoacid toxin PhTx2-6, one of several related neurotoxins in the PhTx2fraction of the P. nigriventer venom (Rezende, 1991; Cordeiro, 1992;Cordeiro, 1995). Optimal alignment of the Acp62F and PhTx2-6 sequences(FIG. 4) shows a 51.7% identity, 82.7% similarity in a region of 28amino acid residues (aas 46-73) of Acp62F to a 27 amino acid region ofPhTx2-6. In this alignment, all six of Acp62F's cysteines in this regionare perfectly aligned with 6 of the 10 cysteines in PhTx2-6 and theother PhTx2 toxins. Furthermore, a seventh cysteine at the N-terminus ofAcp62F is only 3 residues away from a corresponding cysteine in PhTx2-6.

PhTx2 toxins inhibit the closure of voltage-gated sodium channels infrog muscles in vitro, thus prolonging action potentials and increasingmembrane excitability (Araujo, 1993; Cordeiro, 1995). If the sequencesimilarity observed between Acp62F and PhTx2-6 reflects a functionalsimilarity, it could suggest roles for Acp62F in neuromuscular eventsfollowing mating. Such roles could include opening the entry to thesperm storage organs to facilitate efficient storage of transferredsperm or removal of previously stored sperm, delaying movement of thefirst egg through the oviduct to allow sperm time to be stored beforebeing pushed out by the egg or, alternatively, facilitating rapidrelease of eggs following mating.

The toxin-similarity of Acp62F is also intriguing in light of thedemonstrated toxicity of D. melanogaster seminal fluid to mated femaleflies (Chapman, 1995; Rice, 1996). Acp62F enters the hemolymph after itsentry into the mated Drosophila female. Since injection of toxin PhTx2-6into the hemolymph of house flies is lethal (Rezende, 1991; Cordeiro,1995), it is plausible that hemolymph entry of Acp62F could contributeto the decreased lifespan of mated female D. melanogaster (Fowler andPartridge, 1989; Chapman, 1995). Toxicity of seminal fluid is suggestedto be an unintended negative consequence of a seminal fluid componentthat has a reproductively advantageous function (Chapman, 1995; Keller,1995; Chapman and Partridge, 1996). Future genetic analysis of Acp62F isneeded to determine whether this protein plays such a Jekyll-and-Hyderole.

Example 6 Lethality of Acp62F Expressed from a Baculovirus Vector inTrichoplusia ni

The survival of 5^(th) instar Trichoplusia ni was monitored afterinjection with a baculovirus vector carrying the gene encoding Acp62F.The gene encoding Acp62F was cloned into the BacPac vector fromClontech. This vector allows for the introduction and expression of thecloned gene in insect cells. Trichoplusia ni larva were injected withthe baculovirus after reaching the 5^(th) instar stage of development.Two control groups were maintained, uninfected larva and vector injectedlarva. Survival was monitored at 2, 2.5, and 3 days post injection. Theresults shown in FIG. 5 were normalized relative to the survival of thecontrol larva. A significant decrease in larval survival was seen withall the larva infected with baculovirus vectors carrying the Acp62 gene.

Example 7 Lethality of Acp62F Protein in Trichoplusia ni

Acp62F was injected into larvae at the 3^(rd) instar stage ofdevelopment. Survival was monitored over a 12.5 day period afterinjection. Larvae were injected with 115 nano-grams of Acp62F. Controllarvae were injected with an equal molar amount of BSA or an equalvolume of saline solution. The percentage of surviving caterpillars wasdetermined daily. The results are shown in FIG. 6. The percentage ofcaterpillars which pupated is also reported in Table 1. The percentageof pupating animals is virtually identical to the survival of the larvaafter 12.5 days. The injection of Acp62F protein clearly has an effecton the survival of the caterpillars. However, it may not be as effectiveas the introduction of a baculovirus which can continually express theprotein due the half-life of the protein.

TABLE 1 Pupation of Trichoplusia ni After Injection With Acp62F Protein% Pupation Uninjected n = 24 95.8% Saline n = 38 94.9% BSA n = 56 98.2%62FH6 n = 55 78.2%

Example 8 Expression of Acp62F is Lethal to Pre-Adult Fruit Flies

The effect of accessory gland proteins on survival of pre-adult fruitflies was determined by inducing the expression of an accessory glandprotein at different stages in the development of fruit fly larva. Afruit fly which is heterozygous for an accessory gland protein geneunder the control of a heat inducible promoter was bred with a wild-typefly. The resulting progeny were expected to be 50% wild-type and 50%carriers of the heat inducible accessory gland protein gene. Thisallowed for cultures of flies with otherwise identical genetic makeupand at the identical developmental stage. Cultures were heat shocked ona particular day of development and then the flies were allowed to growup (except for the group identified as “all,” which was heat shockedevery day through the first 9 days of development). Normally 80-90% ofeggs produced flies. A similar ratio of eggs developed into flies forthose flies which were produced from flies carrying the Acp 26Aa, 29AB,32CD, 33A, 53E, 63F/64A, and 92EF accessory gland protein genes (SeeFIG. 7).

On the other hand, only half as many flies resulted from eggs producedfrom a fly carrying an inducible Acp62F gene. Furthermore, when theflies were examined it was determined that the homozygous wild-typeflies survived at the expected rates, but that few or no fliesexpressing the Acp62F gene had survived.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

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1. An isolated nucleic acid molecule encoding an accessory gland proteinfrom Drosophila which has the biological property of a serine proteaseinhibitor and includes a serpin signature sequence of SEQ ID NO:
 34. 2.The isolated nucleic acid molecule according to claim 1, wherein thenucleic acid molecule is a deoxyribonucleic acid molecule.
 3. Anexpression vector comprising the nucleic acid molecule according toclaim
 1. 4. A host cell comprising the expression vector according toclaim
 3. 5. The host cell according to claim 4, wherein the host cell isan insect cell.
 6. The isolated nucleic acid molecule that encodes theamino acid sequence of SEQ ID NO: 29 or comprises a nucleotide sequencethat hybridizes over its length to the complement of SEQ ID NO:28 underhybridization conditions comprising hybridization in 0.9M SSC buffer ata temperature of 37° C. and washing with the SSC buffer at 37° C.
 7. Theisolated nucleic acid molecule according to claim 6, wherein theisolated nucleic acid molecule encodes the amino acid sequence accordingto SEQ ID NO:29.
 8. The isolated nucleic acid molecule according toclaim 6, wherein the nucleic acid molecule is a deoxyribonucleic acidmolecule.
 9. The expression vector comprising the nucleic acid moleculeaccording to claim
 6. 10. A host cell comprising the expression vectoraccording to claim
 9. 11. The host cell according to claim 10, whereinthe host cell is an insect cell.
 12. A viral expression vector intowhich has been inserted an nucleic acid molecule encoding an accessorygland protein from Drosophila which has the biological property of aserine protease inhibitor and includes a serpin signature sequence ofSEQ ID NO:
 34. 13. The viral expression vector according to claim 12,wherein the viral vector is a baculovirus vector.
 14. A viral expressionvector into which has been inserted a nucleic acid molecule that encodesthe amino acid sequence of SEQ ID NO: 29 or comprises a nucleotidesequence that hybridizes over its length to the complement of SEQ IDNO:28 under hybridization conditions comprising hybridization in 0.9MSSC buffer at a temperature of 37° C. and washing with the SSC buffer at37° C.
 15. The viral expression vector according to claim 14, whereinthe viral vector is a baculovirus vector.
 16. An isolated proteinencoded by the nucleic acid molecule according to claim
 1. 17. Anisolated Drosophila melanogaster accessory gland protein that has thebiological property of a serine protease inhibitor, wherein said proteinis selected from the group consisting of: a protein having an amino acidsequence according to SEQ ID NO:29; and a protein encoded by a nucleicacid molecule which hybridizes over its length to the complement of SEQID NO: 28 under hybridization conditions comprising hybridization in0.9M SSC buffer at a temperature of 37° C. and washing with the SSCbuffer at 37° C.
 18. The isolated Drosophila melanogaster accessorygland protein according to claim 17, wherein the accessory gland proteincomprises the amino acid sequence according to SEQ ID NO:29.
 19. Anisolated protein encoded by the nucleic acid molecule according to claim6.
 20. The isolated nucleic acid molecule according to claim 6, whereinthe nucleic acid molecule comprises the nucleotide sequence of SEQ IDNO: 28.